No Significant Effects of Lutein, Lycopene or Beta Carotene Supplementation on Biological Markers of Oxidative Stress and LDL Oxidizability in Healthy Adult Subjects

Original Research
No Significant Effects of Lutein, Lycopene or ?-Carotene
Supplementation on Biological Markers of Oxidative
Stress and LDL Oxidizability in Healthy Adult Subjects
Isabelle A. Hininger, PhD, Anita Meyer-Wenger, PhD, Ulrich Moser, PhD, Anthony Wright, PhD, Susan Southon, PhD,
David Thurnham, PhD, Mridula Chopra, PhD, Henk Van Den Berg, PhD, Begona Olmedilla, PhD, Alain E. Favier, PhD,
FACN, Anne-Marie Roussel, PhD, FACN
Laboratoire de Biologie du Stress Oxydant, Faculte´ de Pharmacie, Universite´ Joseph Fourier de Pharmacie, La Tronche,
FRANCE (I.A.H., A.E.F., A.-M.R.), Hoffmann La Roche, Bale, SWITZERLAND (A.M.-W., U.M.), Institute of Food Research,
Norwich, UNITED KINGDOM (A.W., S.S.), University of Ulster, Coleraine, NORTHERN IRELAND (D.T., M.C.), TNO-CIVO
Institute, Zeist, THE NETHERLANDS (H.V.D.B.), Clinica Puerta de Hierro, Madrid, SPAIN (B.O.)
Key words: lutein, lycopene, ?-carotene, LDL oxidizability, oxidative stress, carotenoid supplementation
Objective: The objective of this study was to determine the effect of individual carotenoid supplementation
on biochemical indices of oxidative status in apparently healthy adult males.
Methods:The study was a placebo controlled single blind study. Healthy male volunteers (n?175) were
assigned to four groups. They received daily supplements of ?-carotene (15 mg), lutein (15 mg), lycopene (15
mg) and placebo for three months. The effects of the supplementation on antioxidant status were monitored by
plasma carotenoid, vitamin C and A levels, glutathione (GSH and GSSG) concentrations, protein SH groups,
erythrocyte antioxidant enzyme activities (Cu-Zn SOD, Se-GSH-Px) and susceptibility of LDL to copper-
induced oxidation.
Results:?-carotene, lycopene and lutein supplementation led to significant plasma and LDL increases in
each of these carotenoids, without modifications of other carotenoid levels in plasma or in LDL. The
supplementation failed to enhance the resistance of LDL to oxidation or to modify the LDL polyunsaturated/
saturated fatty acid ratio. Vitamin C, GSH, protein SH groups and antioxidant metalloenzyme activities were also
unchanged.
Conclusion: We did not observe beneficial or adverse effects of lutein, lycopene or ?-carotene supplemen-
tation on biomarkers of oxidative stress. In apparently healthy subjects, carotenoid supplementation does not lead
to significantly measurable improvement in antioxidant defenses.
INTRODUCTION
inversely related to the incidence of cancer [7] and cardiovas-
cular diseases [8]. However, recent interventional trials did not
An increased oxidative stress has been implicated in the
show such a protective effect [9,10]. Most of the reports fo-
incidence of diseases such as cardiovascular disease and cancer
cused on ?-carotene nutriture, but a possible link between
[1,2]. It has been proposed that dietary antioxidants, providing
lycopene intakes and a lower risk of prostate [11], stomach and
protection against free radical attack, could reduce the risk of
pancreatic cancer [12] and myocardial infarction [13] has been
these diseases [3]. A high consumption of fruits and vegetables
also reported. The mechanism of these effects is still not totally
could decrease the risk of cancers [4] or CVD [5], whereas low
explored since carotenoids have multiple biological functions.
intakes of fruits and vegetables increased this risk [6]. Epide-
The protective effect against cardiovascular disease could be
miological data show that carotenoid intakes and status are
related to decreased susceptibility of LDL to oxidation, since
Address reprint requests to: Pr. Anne-Marie Roussel, Laboratoire de Biologie du Stress Oxydant (LBSO), Faculte´ de Pharmacie, UJF, Domaine de la Merci, 38700 La
Tronche, FRANCE. E-mail: Anne-Marie.Roussel@ujf-grenoble.fr
Presented in part as an oral communication at the 38th American College of Nutrition Meeting, New York, October 1997.
Journal of the American College of Nutrition, Vol. 20, No. 3, 232–238 (2001)
Published by the American College of Nutrition
232

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Lutein, Lycopene, ?-Carotene and Oxidative Stress/LDL Oxidizability
the relationship between LDL oxidation and atherogenesis has
Analysis
been well documented [14]. Enrichment of LDL with ?-caro-
Blood was taken from fasting subjects by venipuncture on
tene has been shown to protect LDL against copper-induced
day 0 and after three months of daily supplementation. Plasma
oxidation in vitro [15]. In a previous study [16], we reported the
was separated from cells immediately after collection by cen-
beneficial effects of a carotenoid-rich diet on LDL oxidizability
trifugation at 3,000?g and stored at ?80°C prior to analysis.
in smokers. Other mechanisms of action, such as a sparing
Plasma carotenoid and retinol levels were measured as de-
effect on antioxidant circulating systems (vitamin C, glutathi-
scribed [17]. Plasma ascorbic acid was stabilized and quantified
one or antioxidant enzymes) cannot be totally ruled out [8].
by HPLC [18] in addition to the concurrent quantification of
Considering that supplementation trials with lutein and ly-
copene are scarce, we investigated the effects of lutein and
uric acid which appeared in the same HPLC run. The quality
lycopene supplementation on oxidative stress variables. We
control was assessed through the Fat-Soluble Vitamin QA
measured in healthy male adults, receiving individually lutein,
Program (NIST, USA), and the performance of analysis during
lycopene or ?-carotene, blood biomarkers of oxidative stress
the study was rated by NIST. The concentration of plasma SH
(Vitamin C, Cu-Zn SOD, SeGPx, protein SH groups and GSH/
protein groups was determined in 100 ?L plasma samples [19].
GSSG) and copper-induced LDL oxidizability, in relation to
Erythrocyte Cu-Zn SOD activity was measured after hemoglo-
carotenoid status.
bin precipitation by monitoring the autoxidation of pyrogallol
[20]. Erythrocyte Se-GPx activity was evaluated by the method
of Gunzler [21] using tert-butyl hydroperoxide (Sigma Chem-
ical Co, via Coger, Paris, France) as substrate instead of hy-
METHODS
droperoxide. Results were expressed as nanomoles of NADPH
oxidized per minute per liter. Total glutathione was determined
Subjects
by the modified method of Akerboom [22]. Whole blood (400
?L) was added to 3600 ?L aqueous of metaphosphoric acid
This study was a part of a large interventional European trial
(6% w/v). The mixture was centrifuged for 10 minutes at 4°C.
(AAIR), aimed at investigating the beneficial effects of fruit
The acidic, protein-free supernatants were stored at ?80°C
and vegetable consumption on health. The procedures used
until analysis. Measurement of copper-induced LDL suscepti-
were in accordance to the declaration of Helsinki. The protocol
bility to oxidation was performed as described in De Waart
was approved by all relevant Ethical Committees, and all the
[23], after incubation of LDL isolated from 1 mL of plasma
subjects gave their written informed consent. Adult males 25 to
with a freshly prepared 2.5 mol/L CuCl2 solution in phosphate
45 years old were recruited in five European centers: Grenoble,
buffered saline (PBS), Ph7.4 at 20° for five hours [24], with
France; Coleraine, Northern Ireland; Cork, Ireland; Zeist, Neth-
modification in the isolation step according to Himber [25].
erlands; Madrid, Spain. The volunteers were enrolled all at
Formation of conjugated dienes was measured every 15 min-
once and randomly assigned to four treatment groups at each
utes for five hours by monitoring the increase of the 234 nm
center. They had a stable lifestyle, were apparently healthy,
absorbance following the Cu-induced oxidation of LDL. The
non-smoking and did not take any medications or nutritional
supplements. Exclusion criteria included smoking, serum reti-
length of the lag phase (in minutes) was determined as de-
nol below 1 ?mol/L and BMI?28 kg/m2. Only volunteers
scribed by Frei and Gaziano [26]. Serum cholesterol and trig-
exhibiting normal lipidemic and normal hematological values
lycerides were determined by enzymatic methods. Lipids were
were included. The final number of volunteers at the comple-
extracted with chloroform/methanol, dried under nitrogen,
tion of the study was 175.
transmethylated with methalonic-hydrochloric and separated
by gas chromatography (HP5890A; Hewlett-Packard, Palto
Alto CA) as detailed previously [27]. Briefly, a fused silica
Groups and Supplementation
capillary column (50 mm length, 0.25mm inner diameter, 0.1
mm layer thickness) was used under the following conditions:
All the subjects completed a food-frequency questionnaire
injection at 55°C, 10°C/min from 55–177°C, 1°C/min from
to assess their carotenoid intakes before starting the trial. Sub-
177–218°C, 4°/min from 218 –270°C. C-17 methyl ester was
jects were assigned to four groups receiving 15 mg/day of
used as internal standard, and fatty acids were quantified by
?-carotene (Group 1), lutein (Group 2), lycopene (Group 3) or
using commercial methyl ester standards.
placebo (Group 4) for 12 weeks. ?-carotene and lutein capsules
were a gift from Quest International (Cork, Ireland). Palm oil
carotene (?-carotene [31%], ?-carotene [69%]) and marigold
Statistical analysis
extract were respectively used as the source of ?-carotene and
lutein. Lycopene was a gift from Makhtesim Chemical Wirks
Values are means ?SEM. Values (Week 0 and Week 12)
Ltd (Beer-Sheva, Israel) obtained from natural tomato extract
were compared among groups by analysis of variance
(90% lycopene and 10% ?-carotene).
(ANOVA), with p?0.05 regarded as significant.
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Lutein, Lycopene, ?-Carotene and Oxidative Stress/LDL Oxidizability
Table 1.Characteristics of the Subjects at Baseline1
Group 1
Group 2
Group 3
Group 4
?-carotene n?35
Lutein n?42
Lycopene n?52
Placebo n?46
Age (years)
34.0?1.2
34.2?1.0
32.5?0.8
33.1?1.0
BMI (kg/m2)
24.10?0.6
24.20?0.5
24.40?0.4
24.50?0.5
Cholesterol (mmol/L)
4.78?0.11
5.20?0.14
4.95?0.13
4.99?0.12
Triacylglycerol (mmol/L)
1.07?0.08
1.19?0.08
1.03?0.05
1.06?0.07
1 Mean?SEM.
Table 2. Effects of Supplementation on Plasma Carotenoid Levels1
Plasma Concentrations
Group 1
Group 2
Group 3
Group 4
?mol/L
?-carotene n?35
Lutein n?42
Lycopene n?52
Placebo n?46
?-Carotene
W0
0.39?0.12
0.43?0.12
0.49?0.14
0.47?0.14
W12
1.66?0.13*B
0.49?0.12A
0.66?0.14*A
0.50?0.14A
Lutein
W0
0.22?0.11
0.22?0.12
0.23?0.13
0.21?0.14
W12
0.20?0.11A
0.94?0.13*B
0.23?0.13A
0.20?0.14A
Lycopene
W0
0.62?0.12
0.69?0.13
0.63?0.21
0.69?0.13
W12
0.60?0.12A
0.64?0.12A
1.17?0.15*B
0.69?0.13A
?-Carotene
W0
0.08?0.05
0.07?0.04
0.09?0.02
0.08?0.05
W12
0.79?0.08*B
0.07?0.04A
0.09?0.02A
0.08?0.05A
?-Cryptoxanthin
W0
0.18?0.05
0.18?0.06
0.24?0.04
0.19?0.05
W12
0.15?0.05
0.17?0.05
0.18?0.04
0.2?0.03
Zeaxanthin
W0
0.06?0.02
0.06?0.02
0.07?0.02
0.06?0.02
W12
0.05?0.02A
0.13?0.02*B
0.07?0.02A
0.06?0.02A
1 Mean?SEM.
* Means, within a column, not sharing a common letter are significantly different at p?0.001. AB means, within a row, not sharing a common letter are significantly
different at p?0.05.
RESULTS
lutein and zeaxanthine were enhanced by 327% and 117%,
respectively. In the lycopene group, plasma lycopene increased
At the beginning of the study, the characteristics of volun-
86%. In each group, plasma levels of other carotenoids were
teers in the groups were not statistically different for age, BMI
not altered by the supplementation. On the contrary, we ob-
and lipid profile (Table 1).
served a 35% increase in serum ?-carotene following lycopene
supplementation. Plasma ascorbic acid, retinol and acid uric
were not modified by the supplementation (Table 3).
Effect of the Supplementation on Carotenoid Status
At the onset of the study, ?-carotene, lycopene and lutein
intakes, estimated by food questionnaire, were respectively
Effect of the Supplementation on the Biomarkers of
5.30?0.35 mg/day, 4.84?0.39 mg/day and 2.53?0.19 mg/day.
Oxidative Stress
These intakes were in the normal range according to dietary
recommended intakes for carotenoids. Plasma carotenoid levels
Oxidative stress parameters were not different among the
in the three groups were also in the physiological range [16]
groups at W0, and they remained unchanged after 12 weeks
and not statistically different from those measured in the pla-
(Table 4). As shown in Table 5, for each group there was a
cebo group. After 12 weeks of supplementation (W12), plasma
significant LDL enrichment following carotenoid supplemen-
carotenoid levels increased in each supplemented group, in
tation. LDL susceptibility to oxidation, assessed by lag time
relation to the nature of the carotenoid supplementation (Table
after copper-induced oxidation, was not modified by the sup-
2). In the ?-carotene group, supplementation resulted in an
plementation (Table 5). Moreover, in the four groups, the P/S
increase of 326% in ?-carotene and 887% in ?-carotene, while
ratio, as a potential marker of LDL fatty acid oxidation, re-
plasma retinol remained constant. In the lutein group, plasma
mained unchanged.
234
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Lutein, Lycopene, ?-Carotene and Oxidative Stress/LDL Oxidizability
Table 3. Effect of Supplementation on Plasma Acid Uric, Retinol and Ascorbic Acid1
Group 1
Group 2
Group 3
Group 4
?-carotene n?35
Lutein n?42
Lycopene n?52
Placebo n?46
Uric Acid (?mol/L)
W0
343?10
349?8
340?7
330?9
W12
360?12
357?9
352?9
350?12
Retinol (?mol/L)
W0
2.15?0.07
2.50?0.06
2.15?0.08
2.07?0.07
W12
2.20?0.07
2.18?0.06
2.21?0.08
2.13?0.07
Ascorbic Acid (?mol/L)
W0
52.1?2.7
52.4?2.3
58.2?1.9
56.6?2.3
W12
57.5?3.3
55.9?3.0
55.2?2.8
52.8?3.1
1 Mean?SEM.
Table 4. Effects of Supplementation on Plasma and RBC Antioxidant Defenses1
Group 1
Group 2
Group 3
Group 4
?-carotene n?35
Lutein n?42
Lycopene n?52
Placebo n?46
Se-GSH-Px (U/g Hb)
W0
44.50?1.8
43.0?1.8
41.50?1.9
37.4?1.6
W12
44.90?1.4
45.6?1.8
43.60?1.8
42.3?1.6
Cu,Zn-SOD (U/mg Hb)
W0
1.18?0.0
1.19?0.01
1.21?0.01
1.22?0.01
W12
1.20?0.02
1.18?0.01
1.20?0.02
1.22?0.02
GSH (?mol/L)
W0
977?30
985?25
1022?29
955?29
W12
822?36
868?24
877?32
857?29
GSSG (?mol/L)
W0
30.5?2.4
30.5?2.6
29.1?1.7
28.9?2.1
W12
35.1?2.5
25.9?2.3
28.5?1.9
27.7?2.6
SH groups (?mol/g proteins)
W0
6.49?0.15
6.39?0.11
6.54?0.10
6.32?0.16
W12
6.13?0.09
6.05?0.08
6.06?0.05
6.03?0.07
1 Mean?SEM.
DISCUSSION
?-carotene and ?-carotene were combined in the capsules.
Serum ?-carotene enhancement was higher than that measured
Several studies reported the beneficial effect of ?-carotene
for ?-carotene, in relation to the given doses. We reported a
intakes in decreasing oxidative stress and protecting LDL
similar effect [16], possibly related to a deficient ?-carotene
against oxidation. However, to our knowledge, no studies have
status of the subjects at inclusion. In the lutein group (Group 2),
focused on the effects of lycopene. Our objective was to com-
we also noted a rise of zeaxanthin, as described by others [7].
pare the effects of supplemental intakes of lutein or lycopene to
Lycopene supplementation in Group 3 resulted in an increase in
those of ?-carotene on oxidative stress and especially on LDL
serum lycopene and ?-carotene, in relation to the combined
oxidizability. Carotenoids were given at 15 mg/day, which
composition of the capsule, containing 10% ?-carotene. The
represents relatively high doses, given the nutritional recom-
question of possible adverse effects of carotenoid supplemen-
mendations. In all of the supplemented groups, we observed a
tation on other carotenoid metabolism is a matter of debate.
response to the supplementation leading to a rise in the sup-
Indeed, some studies, using individual or combined doses, have
plementing carotenoid. These data corroborate other results
reported adverse effects of supplemental ?-carotene on plasma
using chemical forms of carotenoids [28 –30], but differ from
levels of other carotenoids [34 –36] or no effect [29]. In males,
trials using a carotenoid-rich fruit and vegetable diet [16],
?-carotene given for six weeks lowered serum lutein levels
which led to moderate increases of carotenoid plasma levels.
[31]. In the Polyp Prevention Study, the supplementation of 25
The discrepancy could be due to a higher bioavailability of
mg/day of ?-carotene for four years did not lead to significant
chemical forms than those of natural sources [31–33]. In this
changes in serum levels of lycopene, ?-carotene, cryptoxanthin
study, we did not observe adverse interactions between caro-
or lutein. In contrast, in the ATBC trial, 20 mg/day of ?-car-
tenoids. These data suggest that the respective absorptions were
otene resulted in increases of ?-carotene, ?-cryptoxanthin and
not modified by a competitive mechanism. In the ?-carotene
lutein [10]. Carotenoids are present in low-density lipoprotein
group, the increase in ?-carotene could be expected since
and they could prevent LDL oxidation [37,38]. After 12 weeks
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION
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Lutein, Lycopene, ?-Carotene and Oxidative Stress/LDL Oxidizability
of supplementation, there was an enrichment of LDL in ?-car-
modified and remained in the physiological ranges. These data
otene, lycopene and lutein, but no modification of oxidizability,
did not agree with a possible sparing mechanism reporting
assessed by lag time and polyunsaturated/saturated fatty acid
interactions between ?-carotene and ascorbic acid in vitro [8].
ratio. Regarding ?-carotene, these data confirm the lack of
In agreement with our study, a previous study [40] did not
protective effects on LDL observed by others [39,40] using
report any change in erythrocyte GSH. In contrast, others [41]
higher doses, but do not corroborate the data observed in our
observed a significant increase in GSH in HIV-infected patients
previous report in smokers [16]. These discrepancies could be
receiving daily high doses of ?-carotene (60 mg/day) for one
due to a better efficiency of the combined carotenoids present
year. However, the low GSH level of the HIV-infected subjects
in food compared to those of an isolated chemical supplement.
in this study, the doses and the duration of the trial could
The complementary effects of all the other antioxidant compo-
explain the discrepancy.
nents of the foods cannot be ruled out. Similar to ?-carotene,
lutein and lycopene did not exhibit properties in enhancing
LDL resistance to oxidation. In this study, we enrolled healthy
CONCLUSION
adults exhibiting an adequate carotenoid status. Our data, fail-
ing to observe a protective effect of supplemental carotenoids
This study showed that in healthy subjects, lutein, lycopene
on LDL oxidation, suggest that such a supplementation in
and ?-carotene supplementation increases greatly the carot-
healthy subjects cannot be recommended as useful. However,
enoid status without adverse biological effects, but does not
they cannot exclude a potential beneficial effect of this supple-
protect against oxidative stress. LDL oxidation, protein oxida-
mentation in protecting LDL from oxidation in deficient sub-
tion and antioxidant enzymatic activities were not modified.
jects.
These data suggest that, when the diet provides adequate ca-
We investigated also the effects of supplementation on
rotenoid intakes, the immediate effects of supplementation are
some circulating markers of oxidative stress. Antioxidant RBC
limited. The consumption of carotenoid-rich foods should be
enzyme activity (Cu-Zn SOD, Se GPx) remained unchanged.
encouraged.
Consistent with previous studies using chemical forms of
?-carotene [41,42] or natural sources [16], we also observed no
effect on antioxidant enzyme activities. A beneficial effect of
ACKNOWLEDGMENT
?-carotene on antioxidant enzyme activities has been reported
after a carotenoid-depleted diet [43,44]. Protein SH group
This research has been supported by the European Union:
concentrations, blood vitamin C and GSH/GSSG were not
AAIR project (AIR-CT93-0888, DG12SSMA).
Table 5. Effect of Supplementation on LDL Oxidizability (min), LDL carotenoid content (ng/mg LDL Cholesterol) and
Polyunsaturated on Saturated Fatty Acid Ratio (P/S)1
Group 1
Group 2
Group 3
Group 4
?-carotene n?35
Lutein n?39
Lycopene n?51
Placebo n?45
Lag Time (min)
W0
95.2?6.02
105?6.56
106?7.37
103.5?6.95
W12
100.7?5.83
93.3?6.49
95.9?7.53
87.9?6.97
LDL?-Carotene
W0
132?1.15
121?1.11
160?1.1
131?1.14
W12
682?1.17*A
179?1.15B
221?1.10B
172?1.13B
LDL Lutein
W0
44?1.11
43?1.10
42?1.07
38?1.10
W12
30?1.22A
121?1.32*B
36?1.13A
24?1.19A
LDL Lycopene
W0
112?1.16
108?1.12
112?1.10
109?1.11
W12
98?1.3A
121?1.10A
177?1.06*B
113?1.10A
LDL Total
(?-Carotene?Lycopene?Lutein)
W0
352?1.11
282?1.10
324?1.09
305?1.11
W12
904?1.11*A
498?1.12B
488?1.07B
317?1.10C
P/S Fatty Acid Ratio
W0
1.88?0.10
1.81?0.07
1.87?0.07
1.80?0.06
W12
1.88?0.17
1.84?0.05
1.85?0.05
1.84?0.09
1 Mean?SEM.
* Means, within a column, not sharing a common letter are significantly different at p?0.001. AB means, within a row, not sharing a common letter are significantly
different at p?0.05.
236
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